Solution phase synthesis of beta-turn peptidomimetic cyclic salts

ABSTRACT

The present disclosure relates to methods of preparing and crystallizing β-turn cyclic peptidomimetic salts of formula I:where R1, R2, R3, R4, R5, R6, R7, R8, R10, X, Y and n are as defined in the specification.The present disclosure provides a more efficient route for preparing a crystalline form of a β-turn cyclic peptidomimetic compounds and salts thereof.

CROSS-REFERENCE

This application is a continuation of Ser. No. 16/184,485, filed Nov. 8,2018, which is a divisional of Ser. No. 15/205,909, filed Jul. 8, 2016,which claims the benefit of priority to U.S. Provisional PatentApplication No. 62/190,596, filed on Jul. 9, 2015. The entire contentsof the foregoing applications are incorporated herein by reference,including all text, tables, and drawings.

FIELD OF THE INVENTION

The disclosure relates to methods of preparing crystalline forms ofβ-turn cyclic peptidomimetic salts.

INTRODUCTION

Considering the promising pharmacological activity of several compoundsin the class of β-turn cyclic peptidomimetic compounds, there exists aneed for the development of a new synthetic methodology that allows forscalability and/or cost effective production.

It is therefore an aspect of this invention to provide new syntheticmethods for the preparation of β-turn cyclic peptidomimetic compounds.It is a further aspect of this invention to provide commercially viablemethods to produce these compounds.

It would be advantageous to develop a method of crystallizing orprecipitating β-turn cyclic peptidomimetic salts to provide an improvedpurification method for these salts. In addition, a crystalline orhighly purified precipitated form of β-turn cyclic peptidomimetic saltswould be useful in formulating pharmaceutical compositions. Thus, thereis a need for methods to produce crystalline or precipitated β-turncyclic peptidomimetic compounds or salts thereof.

SUMMARY OF THE INVENTION

In various embodiments, the invention provides synthetic methods forβ-turn cyclic peptidomimetic salts. Synthetic methods are illustrated inthe embodiments denoted in FIG. 1 (Scheme 1).

Certain embodiments of the present disclosure provide a method ofpreparing a crystalline salt of a β-turn peptidomimetic cyclic compoundof formula (I)

-   -   wherein:        -   R₁ is hydrogen, C₁ to C₆ alkyl, aryl, or an amino acid side            chain substituent of a natural or unnatural amino acid;        -   R₃ is hydrogen, C₁ to C₆ alkyl, aryl, or an amino acid side            chain substituent of a natural or unnatural amino;        -   R₂ and R₄ are independently hydrogen or C₁ to C₆ alkyl, or            R₁ and R₂ together with the carbon atom to which they are            attached form a cyclopropyl, cyclobutyl, cyclopentyl or            cyclohexyl group, or R₁ and R₂, R₃ and R₄ together with the            carbon atom to which they are attached form a cyclopropyl,            cyclobutyl, cyclopentyl or cyclohexyl group;        -   Y is selected from the group consisting of hydrogen, —NO₂,            —COOR₁₄, —OC(R₁₄)₃, —SO₃R₁₄, and —SO₂R₁₄;        -   R₅, R₆, R₇ R₈, and R₉ are independently hydrogen or C₁ to C₆            alkyl;        -   R₁₀ is hydrogen, methyl, t-butyl, or a protecting group; and        -   each R₁₄ is hydrogen, alkyl or aryl;        -   X is selected from the group consisting of O, NR₉, S, P, Se,            C₁ to C₆ alkylene, SO, SO₂ and NH;        -   n is 0, 1, 2, 3, 4 or 5;            the method comprising steps of:    -   (a) providing a protected linear peptidomimetic compound of        formula (IV)

-   -   wherein:        -   R₂, R₄, R₅, R₆ R₇ R₈, and R₁₀ have the meanings defined            above;        -   R₁₁ and R₁₃ are independently hydrogen or a protecting            group;        -   R₁₂ is a protecting group;        -   W₁ and W₃ are independently an amino acid side chain            substituent of a natural or unnatural amino acid, less a            hydrogen atom at the point of attachment to R₁₁ and R₁₃            respectively; and    -   Z is selected from the group consisting of F, Cl, Br and I;    -   (b) selectively deprotecting the compound of formula (IV) to        form a partially protected linear peptidomimetic compound of        formula (III)

-   -   wherein:        -   R₂, R₄, R₅, R₆, R₇ R₈, R₁₀, R₁₁, R₁₃, W₁, W₃, X, Y, Z, and n            have the meanings defined above;    -   (c) cyclizing the partially protected linear peptidomimetic        compound of formula (III) to form a compound of formula (II) by        an intramolecular aromatic nucleophilic substitution reaction

-   -   wherein:        -   R₂, R₄, R₅, R₆, R₇ R₈, R₁₀, R₁₁, R₁₃, W₁, W₃, X, Y, Z, and n            have the meanings defined above;    -   (d) deprotecting an amino acid side chain protecting group in        the compound of formula (II) to obtain the salt of the β-turn        peptidomimetic cyclic compound of formula (I); and    -   (e) crystallizing the β-turn peptidomimetic cyclic compound of        formula (I) to obtain the crystalline salt of formula (I).

In certain embodiments, the disclosure provides a crystalline HCl saltof β-turn peptidomimetic cyclic compound of formula D3 and a method ofpreparing thereof.

DESCRIPTION OF THE DRAWINGS

The drawings illustrate generally, by way of example, but not by way oflimitation, various embodiments discussed in the present document.

FIG. 1 is a general schematic illustration of the synthetic methods forpreparing a crystalline form of a salt of β-turn peptidomimetic cycliccompound of formula (I) according to embodiments of the disclosure.

FIG. 2 is an exemplary reaction scheme for preparing a crystalline formof a HCl salt of β-turn peptidomimetic cyclic compound of structure D3according to an embodiment of the disclosure.

FIG. 3 shows microscope images illustrating the crystal transformationof the crystal of D3 from irregular shape present in a gel toneedle-like crystals present in an acidic solution.

FIG. 4 is a bar chart showing the content of the salt of β-turnpeptidomimetic cyclic compound of formula (I) prepared according toembodiments of the disclosure after treatment with various concentrationof HCl at different time during crystallization.

FIG. 5 is a graph showing the content of an impurity produced during thecrystallization step over time according to an embodiment of thedisclosure.

FIG. 6 is a graph showing the content of another impurity producedduring the crystallization step over time according to an embodiment ofthe disclosure.

FIG. 7 is a graph showing the content of yet another impurity producedduring the crystallization step over time according to an embodiment ofthe disclosure.

FIG. 8 is a diagram of a temperature cycling profile for thecrystallization step according to an embodiment of the disclosure.

FIG. 9 shows microscope images illustrating the appearance of thecrystal of D3 HCl salt from irregular shape present in a gel toneedle-like crystals present in an acidic solution.

FIG. 10 shows microscope images illustrating the crystal transformationof the crystal of D3 HCl salt from crystalline form V to form IV upondrying.

FIG. 11 is a result of the XRPD (X-ray powder difraction) analysis of acrude D3 HCl salt slurry in 0.1M HCl solution.

FIG. 12 is a result of the XRPD analysis of a crude D3 HCl salt slurry0.001 M HCl solution.

FIG. 13A is a result of the XRPD analysis of a crude D3 HCl salt slurryin 0.1M HCl solution after 3 weeks for stability check (top curve), anda reference D3 HCl salt crystalline form V (bottom curve).

FIG. 13B is a result of the XRPD analysis of a reference D3 HCl saltcrystalline form V.

FIG. 14 shows microscope images illustrating the form IV of D3 HCl saltaccording to an embodiment of the disclosure.

FIG. 15 is a result of the XRPD analysis of an amorphous HCl salt of D3sample.

FIG. 16 is a differential scanning calorimetry (DSC) of an amorphous HClsalt of D3.

FIG. 17 is a thermogravimetric analysis (TGA) of the amorphous HCl saltof D3.

FIG. 18 is a dynamic vapor sorption (DVS) of the amorphous HCl salt ofD3, showing mass change as a percentage as a function of time and targetP/Po (i.e., set point relative humidity) as a function of time.

FIG. 19 is a dynamic vapor sorption (DVS) isotherm plots for theamorphous HCl salt of D3 showing change is mass versus target P/Po(i.e., set point relative humidity)

FIG. 20 is a thermogravimetric analysis (TGA) of the amorphous HCl saltof D3 material from the DVS-analysis.

FIG. 21 is a result of the XRPD analysis of an amorphous HCl salt of D3sample before and after DVS.

FIG. 22 is a thermogravimetric analysis (TGA) of the amorphous HCl saltof D3 after equilibrium with water vapor presented in desiccator.

FIG. 23 is a XRPD analysis of the amorphous HCl salt of D3 afterequilibration with water vapor.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the terms “a” or “an” are used, as is common in patentdocuments, to include one or more than one, independent of any otherinstances or usages of “at least one” or “one or more.”

As used herein, the term “about” refers to an amount that isapproximately, nearly, almost, or in the vicinity of being equal to oris equal to a stated amount.

As used herein, the term “crude” refers to a compound or a salt thereofthat is less than 90% pure.

The purity of β-turn peptidomimetic cyclic compound or salt thereof isreferred to by “percent purity.” The measure of purity is not a measureof degree of crystallinity of the crystalline preparation. The puritymay be measured by any means including nuclear magnetic resonance (NMR),gas chromatography/mass spectroscopy (GC/MS), liquid chromatography/massspectroscopy (LC/MS), or high pressure liquid chromatography (HPLC).

A “crystal” refers to one or more crystals of a β-turn peptidomimeticcyclic compound or salt thereof. The determination of a crystal can bedetermined by any means including, optical microscopy, electronmicroscopy, x-ray powder diffraction, solid state nuclear magneticresonance (SSNMR) or polarizing microscopy. Microscopy can be used todetermine the crystal length, diameter, width, size and shape, as wellas whether the crystal exists as a single particle or ispolycrystalline.

A “crystalline” or a “crystalline form” refers to a compound thatcomprises crystals. In the present embodiments, a crystalline or acrystalline form of β-turn peptidomimetic cyclic compound or saltthereof comprises crystals of β-turn peptidomimetic cyclic compound orsalt thereof. In one embodiment, a crystalline β-turn peptidomimeticcyclic compound or salt thereof may comprise some amount of amorphousβ-turn peptidomimetic cyclic compound or salt thereof. In oneembodiment, the crystalline β-turn peptidomimetic cyclic compound orsalt thereof comprises more than 50% by weight of crystals of β-turnpeptidomimetic cyclic compound or salt thereof In another embodiment,the crystalline β-turn peptidomimetic cyclic compound or salt thereofcomprises more than 60%, 70%, 80%, 90% or 95% by weight of crystals ofβ-turn peptidomimetic cyclic compound or salt thereof. The crystallineβ-turn peptidomimetic cyclic compound or salt thereof may comprise50-60%, 60-70%, 70-80%, 80-90% or 90-95% by weight of crystals of β-turnpeptidomimetic cyclic compound or salt thereof. In another embodiment,the crystalline of β-turn peptidomimetic cyclic compound or salt thereofcomprises more than 95% by weight of crystals of β-turn peptidomimeticcyclic compound or salt thereof, e.g., at least 96%, 97%, 98% or 99% byweight of crystals of β-turn peptidomimetic cyclic compound or saltthereof or 100% by weight of crystals of β-turn peptidomimetic cycliccompound or salt thereof.

An “amorphous” form of a β-turn peptidomimetic cyclic compound or saltthereof refers to a β-turn peptidomimetic cyclic compound or saltthereof preparation that comprises few or no crystals of β-turnpeptidomimetic cyclic compound or salt thereof. In one embodiment, anamorphous β-turn peptidomimetic cyclic compound or salt thereofcomprises less than 20%, 10%, 5% or 1% by weight of crystals of β-turnpeptidomimetic cyclic compound or salt thereof.

As used herein, the term “salts” refers to ionic compounds, which mayact as precipitants.

As used herein, the term “pharmaceutically acceptable salt” refers tothe acid addition salt compound formed with a suitable acid selectedfrom an inorganic acid such as hydrochloric acid, hydrobromic acid; oran organic acid such as benzene sulfonic acid, maleic acid, oxalic acid,fumaric acid, succinic acid, p-toluenesulfonic acid and malic acid.

As used herein, the term “unnatural amino acid” refers to all aminoacids which are not natural amino acids as described above. Such aminoacids include the D-isomers of any of the 19 optically active andglycine naturally occurring amino acids described above. Unnatural aminoacids also include homoserine, homocysteine, citrulline,2,3-diaminopropionic acid, hydroxyproline, ornithine, norleucine, andthyroxine. Additional unnatural amino acids are well known to one ofordinary skill in the art. An unnatural amino acid may be a D- orL-isomer. An unnatural amino acid may also be an alpha amino acid, abeta amino acid or a gamma amino acid. An unnatural amino acid may alsobe a post-translationally modified amino acid, such as a phosphorylatedserine, threonine or tyrosine, an acylated lysine, or an alkylatedlysine or arginine. Many forms of post-translationally modified aminoacids are known.

As used herein, the term “protecting group” means that a particularfunctional moiety, e.g., O, S, or N, is temporarily blocked so that areaction can be carried out selectively at another reactive site in amultifunctional compound.

As used herein, the term “protic solvent” refers to a solvent thatcarries hydrogen attached to oxygen as in a hydroxyl group or attachedto nitrogen as in an amine group. Such solvents can donate an H+(proton). Examples of protic solvents include water, ethanol,tert-butanol, and diethylamine.

As used herein, the term “aprotic solvent” refers to a solvent thatcarries few or no hydrogen attached to oxygen as in a hydroxyl group orattached to nitrogen as in an amine group.

As used herein, the term “ring” means a compound whose atoms arearranged in formulas in a cyclic form.

As used herein, the term “alkyl” means a hydrocarbon group that may belinear, cyclic, branched, or a combination thereof having the number ofcarbon atoms designated (i.e., C₁-C₆ means one to six carbon atoms).Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl,n-butyl, t-butyl, isobutyl, sec-butyl, pentyl, iso-amyl, hexyl, octyl,noyl and the like. Alkyl groups may be optionally substituted as definedherein. The term “alkylene,” as used herein, refers to a saturatedaliphatic group derived from a straight or branched chain saturatedhydrocarbon attached at two or more positions, such as methylene(—CH₂—).

As used herein, the term “allyl” refers to compound containing the allylgroup (i.e., CH₂═CH—CH₂—).

As used herein, the term “aryl” means a carbocyclic aromatic systemcontaining one, two or three rings wherein such rings may be attachedtogether in a pendent manner or may be fused. The term “aryl” embracesaromatic radicals such as benzyl, phenyl, naphthyl, anthracenyl,phenanthryl, indanyl, indenyl, annulenyl, azulenyl, tetrahydronaphthyl,and biphenyl. Aryl groups may be optionally substituted as definedherein. The term “arylene” designates any divalent group derived fromaryl (such as above defined) by abstracting a hydrogen atom.

When a group is defined to be “null,” this means that the group isabsent.

When a group is substituted, the substituents may include, withoutlimitation, one or more substituents independently selected from thefollowing groups or a particular designated set of groups, alone or incombination: lower alkyl, lower alkenyl, lower alkynyl, lower alkanoyl,lower heteroalkyl, lower heterocycloalkyl, lower haloalkyl, lower haloalkenyl, lower haloalkynyl, lower perhaloalkyl, lower perhaloalkoxy,lower cycloalkyl, phenyl, aryl, aryloxy, lower alkoxy, lower haloalkoxy,oxo, lower acyloxy, carbonyl, carboxyl, lower alkylcarbonyl, cyano,hydrogen, halogen, hydroxy, amino, lower alkylamino, arylamino, amido,nitro, thiol, lower alkylthio, arylthio, lower alkylsulfinyl, loweralkylsulfonyl, arylsulfinyl, arylsulfonyl, arylthio, sulfonate, sulfonicacid, trisubstituted silyl, N₃, SH, SCH₃, C(O)CH₃, pyridinyl, thiophene,furanyl, lower carbamate, and lower urea.

For purposes of clarity and as an aid in understanding the invention, asdisclosed and claimed herein, the following terms and abbreviations aredefined below:

-   -   AcOH—acetic Acid    -   BAEA—bisaminoethylamine (diethylenetriamine)    -   Boc—t-butyloxycarbonyl    -   tBu—tert-butyl    -   Cbz—benzyloxycarbonyl    -   CTC—chlorotrityl chloride    -   DBU—1,8-Diazobicyclo[5.4.0]undec-7-ene    -   DCM—dichloromethane    -   DIC—1,3-Diisopropylcarbodiimide    -   DIPEA—N,N-diisopropylethylamine    -   DIPE—diisopropyl ether    -   DMF—dimethylformamide    -   EDC—N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide    -   EtOAc—ethyl acetate    -   Fmoc—9-fluorenylmethoxycarbonyl    -   FNBA—2-fluoro-5-nitro-benzoic acid    -   Glu—glutamic acid    -   Gly—glycine    -   HBTU—O-Benzotriazole-N,N,N′,N′-tetramethyl-uronium-hexafluoro-phosphate    -   HSer—homoserine    -   HMPB-MBHA—4-Hydroxymethyl-3-methoxyphenoxybutirric acid MBHA, or        4-(4-hydroxymethyl-3-methoxyphenoxy)-butyryl-p-methyl-benzhydrylamine    -   HOBt—N-hydroxybenzotriazole    -   LPPS—liquid phase peptide synthesis    -   Lys—lysine    -   MeCN—acetonitrile    -   MeTHF—2-methyltetrahydrofuran    -   MTBE—methyl-tert-butyl ether    -   Mtt—methyltrityl    -   Pbf—pentamethyldihydrobenzofuransulfonyl    -   SPPS—solid phase peptide synthesis    -   TBAF—tetrabutylammonium fluoride    -   TBDMS—tert-butyldimethylsilane    -   tBu—tert-Butyl ester    -   TFA—trifluoroacetic acid    -   THF—tetrahydrofuran    -   TIS—triisopropylsilane    -   TMG—tetramethylguanidine    -   Trt—trityl

FIG. 1 shows Scheme 1, depicting general routes to prepare a crystallineform of a salt of a β-turn peptidomimetic cyclic compound of formula(I), including the steps of: (a) providing a protected linearpeptidomimetic compound of formula (IV); (b) selectively deprotectingthe compound of formula (IV) to form a partially protected linearpeptidomimetic compound of formula (III); and (c) cyclizing thepartially protected linear peptidomimetic compound of formula (III) toform a compound of formula (II) by an intramolecular aromaticnucleophilic substitution reaction; and (d) deprotecting an amino acidside chain protecting group in the compound of formula (II) to obtainthe salt of the β-turn peptidomimetic cyclic compound of formula (I);and (e) crystallizing the salt of the β-turn peptidomimetic cycliccompound of formula (I) to obtain the compound of formula (I) incrystalline form.

In certain embodiments, the invention provides a method of preparing acrystalline β-turn peptidomimetic cyclic salt of formula (I) having amacrocyclic ring of from 14 to 16 ring atoms.

In certain embodiments, the method provides compounds where R₁ and R₃are amino acid side-chain substituents. Typically, R₁ and R₃ areindependently derived from natural or unnatural amino acids. Forexample, R₁ and R₃ can independently be derived from the twentynaturally occurring protein amino acids (natural), or modified aminoacids (unnatural), in either enantiomeric configuration. The twentynatural amino acids are alpha-amino acids which include glycine (Gly),alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), lysine(Lys), arginine (Arg), histidine (His), proline (Pro), serine (Ser),threonine (Thr), phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp),aspartic acid (Asp), glutamic acid (Glu), asparagine (Asn), glutamine(Gln), cysteine (Cys) and methionine (Met). The generic structure of analpha-amino acid is illustrated by Formula A: H₂NCH(R*)COOH. R*represents the side chain substituent of the amino acid, which refers toas either R₁ or R₃ in the present disclosure.

An unnatural amino acid typically is any structure having Formula Awherein the R* group is any substituent other than one used in thetwenty natural amino acids. See for instance, Biochemistry by L. Stryer,31(1 ed. 1988), Freeman and Company, New York, for structures of thetwenty natural amino acids. Unnatural amino acids also can be naturallyoccurring compounds other than the twenty alpha-amino acids above.

Such unnatural amino acids include the D-isomers of any of the 19optically active and glycine naturally occurring amino acids describedabove. Unnatural amino acids also include homoserine, homocysteine,2,3-diaminopropionic acid, citrulline, hydroxyproline, ornithine,norleucine, and thyroxine. Additional unnatural amino acids are wellknown to one of ordinary skill in the art. An unnatural amino acid maybe a D- or L-isomer. An unnatural amino acid may also be a beta aminoacid or a gamma amino acid having Formula B: H₂N(CH)_(n)(R*)COOH whereinn is equal to 2 or 3 and R* represents the side chain substituent of anyof the twenty proteinogenic amino acids or any substituent other thanone used in the twenty natural amino acids. An unnatural amino acid mayalso be a post-translationally modified amino acid, such as aphosphorylated serine, threonine or tyrosine, an acylated lysine, or analkylated lysine or arginine. Many forms of post-translationallymodified amino acids are known.

In certain embodiments, the method provides compounds where R₁ and R₃are independently a side chain substituent of two different amino acids.In certain of such embodiments, R₁ and R₃ are independently a side chainsubstituent of lysine, glutamic acid, tyrosine, isoleucine, asparagine,arginine or threonine. In certain embodiments, R₁ and R₃ areindependently a side chain substituent of glutamic acid, lysine,isoleucine or arginine. In one embodiment, R₁ and R₃ are independently aside chain substituent of glutamic acid or lysine. In anotherembodiment, R₁ and R₃ are independently a side chain substituent ofisoleucine or arginine.

In general, the amino acid side-chain substituents (R₁ and R₃) areprotected by suitable protecting groups (R₁₁ and R₁₃, respectively)prior to the cyclization step in the method of preparing β-turn cyclicpeptidomimetic compounds of the disclosure. When the amino acidside-chain substituents, R₁ and R₃, are protected, they are representedas W₁R₁₁ and W₃R₃₃ respectively, where W₁ and W₃ are independently anamino acid side chain substituent of a natural or unnatural amino acid,less one hydrogen atom at the point of attachment to R₁₁ and R₁₃,respectively. The one hydrogen atom is usually found in a functionalgroup such as carboxylic acid, amine, thiol, amide, hydroxyl andguanidine of the amino acid side-chain substituents.

Amino acid side-chain protection of any other sensitive reactive groupsof any molecule involved in the synthesis at any step of the methoddescribed in the disclosure can be achieved by means of conventionalprotecting groups such as those described by T. W. Greene & P. G. M.Wuts (Protective Groups In Organic Synthesis 1991, John Wiley and Sons,New-York); and by Sewald and Jakubke (Peptides: chemistry and Biology,2002, Wiley-VCH, Wheinheim p. 142). For example, alpha amino protectinggroups include, but are not limited to, acyl type protecting groups(e.g., trifluoroacetyl, formyl, acetyl), aliphatic urethane protectinggroups (e.g., t-butyloxycarbonyl (Boc), cyclohexyloxycarbonyl), aromaticurethane type protecting groups (e.g., fluorenyl-9-methoxy-carbonyl(Fmoc), benzyloxycarbonyl (Cbz), Cbz derivatives) and alkyl typeprotecting groups (e.g., triphenyl methyl, benzyl).

Amino acids side chain protecting groups may include tert-butyl etherfor serine, threonine, and tyrosine; Boc for lysine, tryptophan, andhistidine; trityl for serine, threonine asparagine, glutamine, cysteineand histidine; tert-butyl or allyl ester for aspartate and glutamate,Pbf for arginine; benzyl for threonine and serine; Cbz for tyrosine,threonine, serine, arginine, and lysine; alkyl silane for serine andthreonine; and all other protecting groups known in the art.

In certain embodiments, the method provides compounds where R₁₁ and R₁₃are independently selected from the group consisting of trifluoroacetyl,formyl, acetyl, t-butyloxycarbonyl (BOC), cyclohexyloxycarbonyl,fluorenyl-9-methoxy-carbonyl (Fmoc), benzyloxycarbonyl (Cbz), Cbzderivatives, triphenyl, methyl, benzyl, allyloxycarbonyl, tert-butyl,alkyl silane and allyl.

In certain embodiments, the method provides compounds where R₁ is a sidechain substitutent of glutamic acid, and R₁₁ is allyl or tert-butyl. Incertain of such embodiments, R₁ is a side chain substitutent of glutamicacid, and R₁₁ is tert-butyl.

In certain embodiments, the method provides compounds where R₃ is a sidechain substitutent of lysine and R₁₃ is benzyloxycarbonyl,allyloxycarbonyl, or tert-butyloxycarbonyl (BOC). In certain of suchembodiments, R₃ is a side chain substitutent of lysine and R₁₃ istert-butyloxycarbonyl (BOC).

The protecting groups may be removed at a convenient subsequent stageusing methods known in the art. In certain embodiments, the protectinggroups of the amino acid side chains R₁ and R₃ are not removed under thesame condition used to cleave the peptidomimetic compound from the solidsupport. In certain of such embodiments, the protecting groups of theamino acid side chains R₁ and R₃ are not removed under the same acidiccondition used to cleave the peptidomimetic compound from the solidsupport.

In certain embodiments, the method provides compounds where R₂ and R₄are independently hydrogen or C₁ to C₆ alkyl.

In certain embodiments, the method provides compounds where R₅, R₆ andR₇ are hydrogen.

In certain embodiments, the method provides compounds where W₁ and W₃are independently a side chain substitutent of two different aminoacids, less a hydrogen atom on the functional group. In certainembodiments, W₁ and W₃ are independently a side chain substitutent oflysine, glutamic acid, tyrosine, isoleucine, asparagine, arginine orthreonine, less a hydrogen atom on the functional group. In certainembodiments, W₁ and W₃ are independently a side chain substitutent ofglutamic acid or lysine. In certain embodiments, Wi and W₃ areindependently a side chain substitutent of isoleucine or arginine. Incertain embodiments, W₁ is a side chain substitutent of glutamic acid,less a hydrogen atom on the functional group, and R₁₁ is allyl ortert-butyl. In certain embodiments, W₃ is a side chain substitutent oflysine and R₁₃ is benzyloxycarbonyl, allyloxycarbonyl, ortert-butyloxycarbonyl (BOC).

In certain embodiments, the method provides compounds where Y isattached to the benzene ring of the formulas at the meta positionrelative to the point of attachment of the amide group. In certainembodiments, Y is —NO₂.

In certain embodiments, the method provides compounds where Z isattached to the benzene ring of the formulas at the ortho positionrelative to the point of attachment of the amide group. In certainembodiments, Z is F.

In certain embodiments, the method provides compounds where n is 1.

In certain embodiments, the terminal functional group X may be protected(i.e., when R₁₂ is a protecting group). In certain embodiments, when Xis O and R₁₂ is trityl (Trt), tert-butyldimethylsilane (TBDMS), or anyprotecting group that can be removed under conditions that do not causedeprotection of the other protecting groups present in the formula, suchas mild acidic conditions. In certain of such embodiments, when X is Oand R₁₂ is trityl (Trt), or tert-butyldimethylsilane (TBDMS). In certainembodiments, X is S and R₁₂ is trityl. In certain embodiments, X is NHand R₁₂ is 4-methyltrityl (Mtt).

Partial Deprotection

The protecting group R₁₂ may be removed by treating the protected linearpeptidomimetic compound of formula (IV) with a mild acidic solution,such as a solution containing from about 0.01% to about 50% (v/v), from0.1% to 10% (v/v), from 0.5% to 5% (v/v), or from 2% to 5%(v/v) of anacid, such as, trifluoro acetic acid (TFA), or acetic acid (e.g.,50%-95%, or 60%-90% (v/v)). The functional amino acid side chains areprotected using more stable protecting groups that are not cleaved ordeprotected under such mild acidic conditions. Such functional aminoacid side chains can be protected with a strong acid labile protectinggroup on the functional groups. The protecting groups used on thefunctional amino acid side chains are described herein. Thus, theremoval of the protecting group R₁₂ does not cause significantdeprotection of any protected functional group R₁₁ and R₁₃ present informula (IV).

The removal of the protecting group R₁₂ may be carried out in differentsolvent systems. The solvent system is an organic solvent or a mixtureof organic solvents. The solvent system may comprise a polar, aproticsolvent, such as, dichloromethane, acetonitrile or tetrahydrofurane andmixture thereof.

A scavenger can be added to the mild acidic solution to prevent thealkylation of the X group by the alkyl-carbenium ion formed during thereaction. Suitable scavengers include, but are not limited to,triisopropylsilane (TIS), thioanisol, trialkylsilane (e.g.,trimethylsilane, triethylsilane), or mixture thereof. In one embodiment,the scavenger is triisopropylsilane (TIS).

In certain embodiments, the mild acidic solution contains less than 10%,5%, or 3% of a mixture of acid and scavenger. The relative ratio byvolume of acidic material to scavenger in the mild acidic solution usedin the removal of the protecting group R₁₂ can be from about 1:1 toabout 1:5, from about 1:1 to about 1:3, or about 1:2. In one embodiment,the removal of the protecting group R₁₂ is performed in a solutioncomprising less than 10% of a mixture of TFA and TIS in a ratio byvolume of from about 1:1 to about 1:5.

Cyclization

The cyclization reaction may be carried out via an aromatic nucleophilicsubstitution reaction by the nucleophile X.

The cyclization reaction may be performed in polar aprotic solvents,such as, acetonitrile, tetrahydrofurane (THF), dioxanes, or mixturesthereof. In one embodiment, the cyclization reaction is performed inTHF. Significant amounts of solvents like water and methanol are to beavoided as they can act as nucleophiles and interfere with thecyclization. In one embodiment, the cyclization reaction is performed inless than about 0.5% of water, or in the absence of water. In oneembodiment, the cyclization reaction is performed in less than about0.5% of methanol, or in the absence of methanol. In one embodiment, thecyclization reaction is performed in less than about 0.5% of water andin less than about 0.5% of methanol.

In certain embodiments, the cyclization reaction is a base-catalyzedcyclization reaction. The role of the base in the cyclization reactionis to increase the nucleophilic character of the functional group X.Examples of bases that can be used are t-BuOK, C_(s)cO₃, K₂CO₃, ormixtures thereof.

An important aspect of the cyclization step is to control theconcentration of the partially protected linear peptidomimeticintermediate (III) during the reaction to avoid the formation of thedimeric side product. When the cyclization reaction is performed athigher concentrations of the partially protected linear peptidomimeticintermediate (III), e.g., greater than 0.05 M, the rate ofintermolecular reaction increases, and thus accelerates the rate ofdimeric side product formation. Consequently, to avoid the formation ofdimers, the cyclization reaction may be performed at concentrationslower than 0.05 M, particularly, at concentrations lower than 0.03 M, orat concentrations lower than 0.02 M. Such low concentrations (i.e., highdilutions) may be achieved by using large volumes of solvents.Alternatively, the cyclization reaction may be performed by slowaddition of the partially protected linear peptidomimetic intermediate(III) to the reaction media, which can avoid the usage of large volumesof solvent.

The cyclization reaction may be carried out at a temperature from −20°C. to 15° C., from −10° C. to 5° C., or from −8° C. to −1° C. Reactiontime at room temperature can vary from 5 minute to 5 hours according toring size, nucleophile involved in the reaction, base and solvent. Incertain embodiments, the cyclization reaction time at room temperatureis from about 5 minutes to about 1 hour, from about 5 minutes to about30 minutes, or from about 15 minutes to about 20 minutes.

The reaction can be monitored by analytical HPLC, LC-MS or UV as thepartially protected linear peptidomimetic intermediate (III) and theβ-turn peptidomimetic cyclic compound of formula (I) have differentretention time, mass, and UV profiles.

Once the cyclization is completed, an acid solution may be added to thereaction mixture to neutralize the reaction mixture. Suitable acidincludes HCl, KHSO₄, AcOH. For example, an aqueous acid solution may beused. The concentration of the acid solution may vary from about 0.1 Mto about 1 M, or from about 0.05 N to about 0.2 N. Subsequently, theorganic solvent may be removed, e.g., by evaporation, to obtain a cruderesidue slurry.

Alternatively, the crude residue slurry may be filtered to collect thecrude. The residue or filter may be washed with a solvent, e.g., DIPE ormixture thereof.

The crude product can be slurried in EtOAc at 40° C. for 30-40 min,cooled to 0° C., and filtered to collect the product.

Deprotection

For the final deprotection of the amino acids side chain protectinggroups, the protected β-turn peptidomimetic cyclic compound of formula(IV) is treated with appropriate reagents according to the type ofprotecting groups present in the formula. In certain embodiments anacidic solution is used. In some embodiments, the acidic solutionincludes a HCl solution with a concentration of from 10% to 60%, from20% to 50% or from 30% to 40%. HCl can be dissolved in water or inorganic solvents, such as acetonitrile. In one embodiment, the acidicsolution includes a concentrated HCl solution, (e.g., 30-40%) andacetonitrile.

The final deprotection may be performed at a temperature of from about5° C. to about 25° C., from about 10° C. to about 20° C., or from about12° C. to about 17° C. In certain embodiments, the protected β-turnpeptidomimetic cyclic compound of formula (IV) may be suspended in anacidic solution at a temperature of from about 5° C. to about 25° C.,from about 10° C. to about 20° C., or from about 12° C. to about 17° C.(e.g., at about 15° C.).

Crystallization

In certain embodiments, the β-turn peptidomimetic cyclic compound offormula (I) may be crystallized or precipitated via abasification-acidification method. The basification-acidification methodincludes the steps of: contacting the compound or salt of formula (I)with a basic solution in water having a basic pH, and reducing the pH toobtain an acidic pH to precipitate or crystallize the compound or saltof formula (I).

The compound of formula (I) may be treated with an aqueous basicsolution containing an inorganic base, such as NaOH, LiOH, KOH, Mg(OH)₂or mixtures thereof. The basic solution may be heated to a temperatureof from about 30° C. to about 70° C., or from about 40° C. to about 60°C. The concentration of the basic solution may be from about 0.1 M toabout 5 M, from about 1 M to about 3 M, or from about 1.5 M to about 2M. Typically, from about 2 to about 8 equivalents, from about 3 to about7 equivalents, or from about 3 to about 5 equivalents of the inorganicbase relative to the crude final product (i.e., formula (I)) may beused. The basic pH may be from about 9 to about 12, from about 9 toabout 11, from about 8 to about 12.

The method may further include a step of filtering the basic solutioncontaining the compound of formula (I) prior to reducing the pH of thebasic solution. The purpose of the filtering is to remove any insolublematerials. The filtering may be performed at a temperature above roomtemperature, e.g., from about 40° C. to about 70° C., or from about 45°C. to about 60° C.

The filtrate or the basic solution mixture may be acidified by admixingthe basic solution with an acid to achieve an acidic pH of from about 0to about 4, from about 0 to about 3, or from about 0 to about 2. Aninorganic acid may be used to acidify the basic solution. Examples ofinorganic acid include, but are not limited to HCl, HBr, nitric acid,sulfuric acid, phosphoric acid or mixtures thereof. Typically, fromabout 3 to about 9 equivalents, from about 4 to about 8 equivalents, orfrom about 5 to about 7 equivalents of the acid may be used relative tothe crude final product of formula (I).

The crystals of the salt of form (V) can be formed or precipitated fromthe acidified solution. The acidic pH may promote the formation of agel, subsequently transforming into a slurry/cloudy solution thatcontains crystals. Too less acid may slow down the transformation fromgel to slurry. Typically, from about 3 to 7 equivalents, from about 4 to6 equivalents, or about 5 equivalents of acid relative to the crudefinal product (i.e., formula (I)) may be used. In certain embodiments, aminimum of 2 equivalents excess of acid relative to base is required topromote the crystallization of the compound of form (IV). Once theneedles crystals (form V) are formed, they are stable in the slurry andcan be stored in room temperature for at least 3 weeks.

To facilitate the growth or precipitation of crystals with the minimalamount of impurity formation, the acidified solution may be agitated orstirred for a period of time and/or kept under a temperature of fromabout 0° C. to about 60° C. During the crystallization process, samplesmay be taken from the acidified solution and monitored for crystal orprecipitate formation by microscopic examination and the yield may befollowed spectrophotometrically (e.g., HPLC). To avoid formation ofimpurities and to facilitate growth or precipitation of crystals duringthe formation of crystals, a temperature profile may be used duringcrystallization. That is, the acidified solution may be agitated underone or more temperature ranges each for a specific period of time. Forexample, the one or more temperature may include a first temperature, asecond temperature, a third temperature and so forth, and eachtemperature can be chosen, independently, from any of the followingtemperature ranges: from about 0° C. to about 30° C., from about 0° C.to about 20° C., from about 0° C. to about 10° C., from about 10° C. toabout 40° C., from about 10° C. to about 30° C., from about 10° C. toabout 20° C., from about 20° C. to about 50° C., from about 20° C. toabout 40° C., from about 20° C. to about 30° C., from about 30° C. toabout 60° C., from about 30° C. to about 50° C., from about 30° C. toabout 40° C., from about 40° C. to about 70° C., from about 40° C. toabout 60° C., from about 40° C. to about 50° C., from about 50° C. toabout 70° C., from about 50° C. to about 60° C., etc. The acidifiedsolution may be agitated for a period of time at each temperature range,and each period of time can be chosen, independently, from any of thefollowing time ranges: for at least 5, 10, 15, or 60 minutes or forduration of 1, 2, 3 hours, and so forth, and up to 1 month. In certainembodiments, the agitation period of time may be from about 5 minutes toabout 20 hours, from about 15 minutes to about 15 hours, from about 30minutes to about 10 hours, from about 1 hour to about 5 hours, fromabout 2 hours to about 4 hours, etc.

The method of the disclosure may produce in a yield of at least about 30mole %, or from about 35 to about 45 mole %. These yields are based onthe moles of the limiting reactant, for instance, the moles of the HSercompound.

In one specific aspect, the disclosure provides a crystalline HCl saltof β-turn peptidomimetic cyclic compound of formula D3, also referred toherein simply as “D3” and the method of preparing thereof. The structureof the HCl salt of D3 is shown below:

FIG. 2 shows exemplary reaction scheme (Scheme 2) which depicts a routeto prepare a crystalline HCl salt of the β-turn peptidomimetic cycliccompound of formula D3.

In embodiments, the present disclosure provides a method of preparing acrystalline form of a HCl salt of a β-turn peptidomimetic cycliccompound having the following structure:

-   -   the method comprising steps of:    -   (a) providing a protected linear peptidomimetic compound of        formula (4a);

-   -   (b) selectively deprotecting the compound of formula (4a) to        form a partially protected linear peptidomimetic compound of        formula (3a);

-   -   (c) cyclizing the partially protected linear peptidomimetic        compound of formula (3a) to form a compound of formula (2a) by        an intramolecular aromatic nucleophilic substitution reaction;

-   -   (d) deprotecting an amino acid side chain protecting group in        the compound of formula    -   (II) to obtain HCl salt of D3; and    -   (e) crystallizing HCl salt of D3 to obtain the HCl salt of D3 in        crystalline form.

In embodiments, the protected linear peptidomimetic compound of formula(4a) may be obtained from a liquid phase peptide synthesis processcomprising the step of coupling Fmoc-Hser(Trt)-OH with H-Gly-OtBu·HClthereby forming a dipeptide Fmoc-Hser(Trt)-Gly-OtBu. The process mayfurther comprise the step of coupling the dipeptide H-Hser(Trt)-Gly-OtBuwith Fmoc-Lys(Boc)-OH thereby forming a tripeptideFmoc-Lys(Boc)-Hser(Trt)-Gly-OtBu. The process may further comprise thestep of coupling the tripeptide H-Lys(Boc)-Hser(Trt)-Gly-OtBu withFmoc=Glu(Otbu)-OH thereby forming a tetrapeptideFmoc-Glu(OtBu)-Lys(Boc)-Lys(Boc)-Hser(Trt)-Gly-OtBu.

One of more of the coupling steps may be performed in the presence ofdiisopropylamine (DIPEA) and a condensation reagent, such asO-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyl-uronium-tetrafluoroborat-e(HATU), 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride(EDC·HCl),O-benzotriazol-N,N,N′,N′-tetramethyl-uronium-hexafluoro-phosphate(HBTU), 1-hydroxybenzotriazole (HOBt),benzotriazol-1-yl-oxy-tris-pyrrolidino-phosphoniumhexafluoro phosphate(PyBOP), 1-hydroxy-7-azabenzo triazole, TBTU ((Benzotriazolyl)tetramethyluronium tetrafluoroborate), TATU ((7-Azabenzotriazolyl)tetramethyluronium tetrafluoroborate) and COMU((1-Cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbeniumhexafluorophosphate), Oxyma [ethyl 2-cyano-2-(hydroxyimino)acetate],K-Oxyma (2-Cyano-2-(hydroxyimino)acetic acid ethyl ester, potassiumsalt) or mixtures thereof. In one embodiment, the condensation reagentis EDC·HCl/HOBt. The coupling step of Fmoc-Hser(Trt)-OH withH-Gly-OtBu·HCl may be performed in the presence of DIPEA andEDC·HCl/HOBt. The coupling step of the dipeptide H-Hser(Trt)-Gly-OtBuwith Fmoc-Lys(Boc)-OH may be performed in the presence of DIPEA andEDC·HCl/HOBt. The coupling step of the tripeptideH-Lys(Boc)-Hser(Trt)-Gly-OtBu with Fmoc-Glu(Otbu)-OH may be performed inthe presence of DIPEA and EDC·HCl/HOBt.

The step of removing the N-terminal Fmoc protecting group of any of thepeptides may be performed in the presence of BAEA to provide anN-terminal free amino group.

In certain embodiments, the crystalline form of the disclosure comprisescrystalline form IV, form V or mixtures thereof. These crystalline formscan be produced by the methods described herein and are substantiallyfree of other crystalline forms (i.e., other than form IV or form V).The term “substantially free” refers to an amount of 10% or less ofanother form, for example 8%, 5%, 4%, 3%, 2%, 1%, 0.5%, or less ofanother form. The crystalline form of the disclosure can becharacterized by X-Ray powder diffraction (XRPD), thermal data bydifferential scanning calorimeter (DSC) and thermal gravimetric analysis(TGA), dynamic vapor sorption (DVS), and/or gravimetric vapor sorption(GVS).

In one embodiment, the disclosure provides a crystalline form IV of aHCl salt of the β-turn peptidomimetic cyclic compound of formula D3. Inone embodiment, the disclosure provides a crystalline form V of a HClsalt of the β-turn peptidomimetic cyclic compound of formula D3. In oneembodiment, the disclosure provides a crystalline form including amixture of form IV and form V of a HCl salt of the β-turn peptidomimeticcyclic compound of formula D3.

In certain embodiments, the form IV is hygroscopic. In certainembodiments, the crystalline form IV may have a weight increase of fromabout 10% to about 30% at relative humidity (RH) from 60% to 100%, wherethe weight increase is due to the weight gain of water content. Incertain embodiments, the absorption or desorption of water process inthe crystalline form may be reversible. In certain embodiments, thecrystalline form IV has an irregular shape. In certain embodiments, thecrystalline form V is in needle shaped form.

The transformation of the crystalline forms (between form IV and form V)of the disclosure occurs when varying RH. For example, form IV may betransformed to form V when exposed to high relative humidity, such asfrom 75% to 100%, from 85% to 100%, or from 95% to 100% RH. Form IV maybe transformed to form V when treated with an acidic solution, e.g., anaqueous solution of hydrochloric acid, where the concentration ofhydrochloride acid from about 0.001M to about 0.5M, from about 0.01M toabout 0.2M, from about 0.05M to about 0.1M. Form IV may be transformedto a mixture of form V and some other forms when treated in a verydilute acidic solution, e.g., having a concentration of lower than0.001M, or pure water (i.e., in the absence of acid or base). Form V maybe transformed back to form IV upon drying. The rate of thetransformation may be dependent on the concentration of the acidicsolution. Generally, the rate of transformation from form V to form IVis faster when the crystalline form V is treated with a higherconcentration of acidic solution.

In certain embodiments, the crystalline salt of the β-turnpeptidomimetic cyclic compound of formula D3 contains needle shapedcrystals.

In certain embodiments, the crystalline form of a salt of a β-turnpeptidomimetic cyclic compound of formula D3 is obtained from a solutioncontaining at least 60% water.

In certain embodiments, the crystalline form of a salt of a β-turnpeptidomimetic cyclic compound of formula D3 is a hydrate.

In certain embodiments, the crystalline form of the HCl salt of D3 ischaracterized by an XRPD pattern with characteristic peaks atdiffraction angles (° 2theta) of 6.7±0.2 and 9.1±0.2. In one embodimentthe crystalline form of the HCl salt of D3 is characterized by an XRPDpattern with characteristic peaks at diffraction angles (° 2theta) of6.7±0.2, 9.1±0.2, 4.4±0.2, 5.1±0.2 and 2.6±0.2.

In one embodiment the crystalline form of the HCl salt of D3 ischaracterized by an XRPD pattern with having two or more, three or more,four or more, or five or more characteristic peaks at diffraction angles(° 2theta) selected from 6.7±0.2, 9.1±0.2, 4.4±0.2, 5.1±0.2, 2.6±0.2,11.5±0.2, 15.3±0.2, 16.6±0.2, 17.7±0.2, 18.2±0.2, 20.2±0.2, 21.6±0.2,22.1±0.2, 22.5±0.2, 23.2±0.2, 24.1±0.2.

In one embodiment of this aspect, there is provided a crystal form ofwhich has a powder X-ray diffraction pattern essentially as set out inFIG. 13B.

EXAMPLES

The following examples are merely illustrative of the disclosure andshould not be considered limiting the scope of the invention in any way,as these examples and other equivalents thereof will become apparent tothose skilled in the art in light of the present disclosure and theaccompanying claims. All percentages used in the application are percentweight by weight (w/w) unless otherwise noted.

EXAMPLE 1

FIG. 2 (Scheme 2) illustrates the synthesis of a HCl salt of D3, whichis one embodiment of β-turn peptidomimetic cyclic compound of formula(I).

Synthesis of Compound 4a can be carried out by standard stepwise LPPS(liquid phase peptide synthesis) procedures. See for example, L. A.Carpino et al., Organic Process Research & Development 2003, 7, 28-37)

(a) Synthesis of dipeptide H-Hser(Trt)-Gly-OtBu

To a vessel containing Fmoc-Hser(Trt)OH (37.5 g), HCl×H-GlyOtBu (12 g)and HOBt (10.8 g) were added EtOAc (227 ml) and DMF (45 ml). The mixturewas agitated at 5° C. DIPEA (12.5 ml) and EDC×HCl (15 g) were added. Themixture was agitated at 20° C. to complete conversion (normally <2 h).On completion, the reaction mixture was washed twice with NaCl (23%, 113ml). It was made sure that no aqueous phase was left behind in thereactor, and that no part of the organic layer was discarded afterwashings.

To the organic solution were added DMF (45 ml) and BAEA (34.5 ml). Themixture was agitated at 20° C. for 30 min for complete removal of theFmoc group. On completion, the reaction mixture was extracted with NaCl(23%, 113 ml) once, and with NaHCO₃ (4.8%, 113 ml) twice. The organicsolution was ready for next coupling. It was made sure that no aqueousphase was left behind in the reactor, and that no part of the organiclayer was discarded after washings.

(b) Synthesis of tripeptide H-Lys(Boc)-Hser(Trt)-GlyOtBu

The organic solution containing the dipeptide was diluted with DMF (227ml) and stirred at 5° C. To this solution were added Fmoc-Lys(Boc)OH (30g), HOBt (10.8 g), and EDC×HCl (15 g) and EtOAc (45 ml). The mixture wasagitated at 20° C. to complete conversion (normally <1 h). Oncompletion, BAEA (34.5 ml) was added and the mixture was agitated for 30min for complete removal of the Fmoc group. On completion, the reactionmixture was washed with a mixture of NaCl (23%, 170 ml) and water (150ml) once, and with NaHCO₃ (4.8%, 113 ml) twice. The organic layer wasdiluted with EtOAc (30 ml), and was ready for the next coupling. It wasmade sure that no aqueous phase was left behind in the reactor, and thatno part of the organic layer was discarded after washings.

(c) Synthesis of tetrapeptide H-Glu(OtBu)-Lys(Boc)-Hser(Trt)-GlyOtBu

The organic solution containing the tripeptide was diluted with DMF (227ml) and stirred at 5° C. To this solution were added Fmoc-Glu(OtBu)OH(28.5 g), HOBt(10.8 g), and EDC×HCl (15 g). The mixture was agitated at20° C. to complete conversion (normally <2 h). On completion, BAEA (34.5ml) was added and the mixture was agitated for 30 min for completeremoval of the Fmoc group. On completion, the reaction mixture waswashed with a mixture of NaCl (23%, 113 ml) and water (49 ml). Theorganic solution was diluted with DMF (169 ml) and EtOAc (113 ml), andthen heated to 40° C. The solution was washed with a mixture of NaCl(23%, 113 ml) and water (38 ml) at 40° C. After phase separation, theorganic layer was washed with a mixture of NaCl (23%, 56 ml) and water(56 ml) at 40° C. The pH of the aqeuous layer was adjusted to pH 5-5.5(indicator sticks) with KHSO₄ (1 M) and the phases were separated. Theorganic solution was ready for next coupling. It was made sure that noaqueous phase was left behind in the reactor, and that no part of theorganic layer was discarded after washings.

(d) Synthesis of tetrapeptideFNBA-Glu(OtBu)-Lys(Boc)-HSer(Trt)-Gly-OtBu, Compound 4a

The organic solution containing the tetrapeptide was diluted with DMF(227 ml) and stirred at 5° C. To this solution were added FNBA (14.3 g),EDC×HCl (15 g) and DIPEA (13.4 ml). The mixture was agitated at 20° C.to complete conversion (normally <5 h). On completion, the reactionmixture was stirred with MeTHF (450 ml), NaCl (23%, 113 ml) and NaHCO₃(4.8%, 113 ml). The layers were separated and the organic layer washeated to 40° C. and subsequently washed at this temperature with NaHCO₃(4.8%, 226 ml), and finally with water (226 ml). It was made sure thatno aqueous phase was left behind in the reactor, and that no part of theorganic layer was discarded after washings.

The organic layer was concentrated to ca 226 ml at 40° C. under vacuum.To the residue was added MeTHF (226 ml), and then concentration to ca226 ml was repeated. To the residue were added MeTHF (226 ml) and MTBE(450 ml), and the slurry agitated at 20° C. for 5 h. The solid productwas collected on a filter and washed with MTBE (226 ml) twice and driedat 20° C. under vacuum. Dry product, compound 4a (55.6 g) was obtained,yield: 85.5% with assay correction. HPLC: 90.6%, dibenzofulvene: 9%.

(e) Partial Deprotection of the pentapeptide (de-tritylation), Compound3a

The pentapeptide (55.2 g) was added in 5-7 portions to a mixture ofacetic acid (450 ml) and water (50 ml) at 40° C. After addition, thewhite suspension was agitated for 7-9 h to complete conversion (>98%).On completion, the reaction mixture was cooled to 20° C. and thenextracted with a mixture of MTBE (552 ml), NaCl (23%, 276 ml) and water(110 ml). The organic layer was extracted twice with KHSO₄ (1M, 276 ml)at 20° C., three times with NaHCO₃ (4.8%, 276 ml) at 40° C. , and oncewith water (276 ml). It was made sure that no aqueous phase was leftbehind in the reactor, and that no part of the organic layer wasdiscarded after washings. The organic layer was cooled to 20° C. andagitated overnight to allow the product to precipitate. Heptane (552 ml)was added to the slurry, which was agitated for 1 h (more productprecipitated). The solid product was collected on a filter and washedtwice with heptane (2×276 ml) and twice with DIPE (2×414 ml). Afterdrying (52° C., 4 h) in vacuo, 39.4 g of the desired partiallydeprotected linear peptide, compound 3a, was obtained, yield: 93%without assay correction.

(f) Cyclization

A slurry of t-BuOK (8.13 g) in THF (196 ml) was added to a cold (−8 to−1° C.) solution of the partially deprotected linear peptide (39.4 g) inTHF (1560 ml). The mixture was agitated for 15-20 min (inner temperaturerose to 4° C.) to obtain complete conversion. On completion, aqueous HCl(0.1 N, 189 ml) was added to the reaction mixture. THF was removed byvacuum evaporation at 40° C. The residue was diluted with water (200 ml)and agitated for ca 3 min. The solid crude product was collected on afilter, washed twice with water (2×200 ml), twice with DIPE (2×200m1),and dried at 45° C. under vacuum for 2 h. The dried crude product (36.1g) was suspended in EtOAc (788 ml). The suspension was agitated at 40°C. for ca 40 min. and then cooled to 0° C. and kept agitated for 2 h.The solid product, compound 2a, was collected on a filter and washedtwice with DIPE (2×200 ml) on the filter. After drying, 34.6 g of thepurified cyclic peptide was obtained, yield: 78.6% with assaycorrection, HPLC 96.8%.

(g) Final Deprotection

The purified cyclic peptide (33.3 g) obtained from the cyclization stepwas suspended in a mixture of MeCN (330 ml) and concentrated HCl (37%,54 ml). The suspension was agitated at 15° C. for 1 h. The suspensionturned to a solution and then to a slurry again. On completion, MeCN(330 ml) was added and the mixture was agitated for 1 h. The crudeproduct, HCl salt of D3, was collected on a filter, washed twice withMeCN (2×330 ml), and dried at 20° C. under vacuum. Yield: 24.5 g.

(h) Crystallization

Basification-Acidification Method:

The crude product (24.5 g) was suspended in water (245 ml) and heated to50° C. To this suspension (may appear as a gel) was added NaOH (2 M,47.6 ml). The mixture was stirred at 50° C. for 1 h until a solution wasobtained (may be cloudy). This solution was filtered hot to remove anyinsoluble materials. The vessel was rinsed with water (74 ml), therinsing liquid was passed through the filter, and filtrates werecombined. The combined filtrates were transferred to another vessel, andheated to 50° C. with efficient agitation. To this warm solution wasadded HCl (4 M, 47.6 ml). A gel was formed initially. The agitation wascontinued at 50° C. for 40 min, at 40° C. for 50 min, at 30° C. for ca.14 h, and at 1° C. for 8 h to obtain a white slurry. The purified finalproduct (white needles) was collected on a filter, washed with HCl (1 M,50 ml) and dried at 20° C. under vacuum. Yield: 21 g, assay: 85.66%.HPLC: 98.9%. Overall yield: 53.6% with assay correction starting fromFmoc-Hser(Trt)OH.

The final product tended to from a gel at pH 2-8, but at pH>9 it formeda stable solution appropriate for clear filtration. After acidificationof the clear solution with 37% HCl to pH≥0, the final product HCl saltformed a gel first and then the gel was transformed to fine needles(see, FIG. 3 ) of high purity.

EXAMPLE 2 Optimization of the Crystallization Process

A series of experiments were performed to avoid formation of impuritiesduring the crystallization and to obtain a robust process at largerscale. The temperature and the strength of HCl were varied andsummarized in Table 1 below. A sample was removed from each experimentat a certain time: 2 h, 4 h, 8 h and 22 h. The content of the finalproduct and the three major impurities in the samples were monitoredwith HPLC (FIGS. 4-7 ).

TABLE 1 40° C. 50° C. 60° C. 5 equiv. HC1 A B C 7 equiv. HC1 D E F

FIG. 4 is a bar chart showing the HPLC percent area of the final productat different conditions (i.e., at various temperature and concentrationof HCl (labeled as A, B, C, D, E and F)) at 2 hours, 4 hours, 8 hoursand 22 hours during crystallization. The starting purity was 96.2%.

FIG. 5 is a graph showing the formation of impurity Des-Gly duringcrystallization over time a period of 22 hours. The followingconclusions can be made: (a) at 40° C., the final product is stable inthe presence of 5 and 7 equiv. of HCl for 22 hours; (b) at 50° C., thefinal product is stable in the presence of 5 and 7 equiv. HCl for 4hours; (c) at 60° C., the final product starts to degrade after 2 h.

FIG. 6 is a graph showing the formation of an unknown impurity duringcrystallization over time a period of 12 hours. The impurity was elutedat 0.93 relative retention time (RRT 0.93) measured by the HPLC. Thefollowing conclusions can be made: (a) at 40° C., the formation of RT0.93 is very slow regardless if it is added 5 equiv. or 7 equiv. HCl;(b) at 50° C., the formation of RT 0.93 is slightly higher than that at40° C. within 8 hours. It is accelerated after 8 h, and more RT 0.93 isgenerated using 7 equiv. HCl compared to 5 equiv. HCl; (c) at 60° C.,the formation of RT 0.93 is increased rapidly.

FIG. 7 is a graph showing the formation of an unknown impurity duringcrystallization over time a period of 12 hours. The impurity was elutedat 1.11 relative retention time (RRT 1.11) measured by the HPLC. Thefollowing conclusions can be made: (a) at 40° C., the formation of RRT1.11 is very slow regardless if 5 equiv. or 7 equiv. HCl are added; (b)at 50° C., the formation of RRT 1.11 is slightly higher than at 40° C.within 4 hours. It is accelerated after 4 h, and more of the unknownimpurity is generated using 7 equiv. HCl than using 5 equiv. HCl; (c) at60° C., the formation of RRT 1.11 is increased rapidly.

EXAMPLE 3 Temperature Cycling During Crystallization

In order to understand the crystallization process, the temperature wascycled during the crystallization by lowering it from 50° C. to 20° C.,and then raising it to 50° C. and lowering to 20° C. again. Thetemperature cycling diagram is shown in FIG. 8 . Samples were removedfrom the slurry at intervals and analyzed by microscopy. FIG. 9 showsthe crystal transformation of the final product under microscopeanalysis.

A gel was transformed to a crystalline (needles) slurry after 14 hoursat 40° C. These needles were not damaged during the temperature cyclingfor totally 47 hours, including long cooling at 5° C. This means thatthis crystalline form was stable under these conditions and thetransformation required a long time. Throughout development of thecrystallization method several experiments indicated that the purity ofthe crude final product influences the transformation rate from gel tocrystalline material. After crystallization, the purity of thecrystalline product was above 97% (e.g., 97.2% -99.1%) measured by HPLC.The crystalline product was easier to filter than the gel, although thefiltration time was relatively long. After drying, the needles weretransformed to an irregular solid, with some crystallinity. FIG. 10shows the crystal transformation on the filter under microscopeanalysis.

EXAMPLE 4 Studies of Transformation of Crystal Forms

A crude batch of D3 sample was slurried in 0.1M HCl solution, 0.001M HClsolution to study possible transformations. Approximately 60 mg of crudeD3 sample was slurried in 0.6 ml solution. The slurry was left withmagnetic stifling at room temperature. In the 0.1M HCl slurry, needleswere observed after 1-2 days. The slurry was analyzed by XRPD on aporous plate, see FIG. 11 . The crystal form corresponds to form V,i.e., a fast transformation from form IV to form V occurs. In purewater, the transformation was much slower and an analysis was made after2 weeks. The slurry includes mainly form V but there are some extrapeaks which may indicate the presence of other forms. It should benoticed that the crude material may have contained excess amounts of HClwhich influences the pH. The pH in the pure water slurry wasapproximately 3.

In 0.001M HCl solution, the transformation was also slow and theanalysis was made after 2 weeks. The XRPD, see FIG. 12 , shows thatcrystalline material had formed. There are a lot of similarities withthe pure water (i.e., 100% water) slurry, i.e., it seems to be a mixtureof form V and some other form.

The 0.1M HCl slurry was analysed a second time after 3 weeks. See FIG.13A. It is still corresponding to form V, i.e., the slurry seems verystable with respect to polymorphic form.

EXAMPLE 5 Characterization of the Amorphous Form of D3

FIG. 14 shows microscope pictures of a dry sample of an amorphous HClsalt of D3, which was crystallized but lost its crystallinity upondrying which was obtained prior to crystallization.

The amorphous HCl salt of D3 sample was analyzed by XRPD, see FIG. 15 .It can be concluded that the material is form IV although it is mainlyamorphous. The peak at 2 Theta=32° corresponds to sodium chloride in thematerial.

The DSC of the amorphous HCl salt of D3 is shown in FIG. 16 . There is asmall endotherm around 60° C., a week exothermic event around 180-190°C. followed by an endotherm around 220° C. Since form IV is mainlyamorphous it is expected that the thermal events are smaller and notwell defined.

The thermogravimetric analysis (TGA) of the amorphous HCl salt of D3 isshown in FIG. 17 , which shows a weight loss of approximately 1.7% up to100-110° C.

The amorphous HCl salt of D3 sample was analyzed by dynamic vaporsorption (DVS), see FIG. 18 . There is a continuous uptake of water upto 95% relative humidity corresponding to a weight increase of 32%. At20% RH (relative humidity), the change in mass is 4%, at 40% RHapproximately 7%. Despite the large uptake of water, the process seemscompletely reversible and there is almost no hysteresis effect, i.e.,the uptake and loss of weight is almost identical, see FIG. 19 . It isnoted that both cycles are almost identical.

The amorphous HCl salt of D3 sample after the DVS-analysis was analyzedwith TGA, see FIG. 20 . There is a weight loss of 3.9%, whichcorresponds well with the water uptake at RH=20%.

The amorphous HCl salt of D3 sample was analyzed again by XRPD, see FIG.21 , which shows that the material was still very similar to thestarting material.

In order to study the behavior at very high RH (near 100%) an amorphousHCl salt of D3 sample was added to a microscope slide and put in adesiccator saturated with water vapor. The sample was left overnight andthen quickly analyzed by TGA, see FIG. 22 . The TGA analysis showed awater loss of approximately 30%. It should be noticed that the samplewas still in solid form, i.e., there is no deliquescence. The sample wasalso analyzed by XRPD, see FIG. 23 . The analysis shows that the sampleis more crystalline than the starting material (FIG. 21 ) and there is agreat similarity with form V. The peaks of the reference form V areshifted towards smaller 2 Theta values, i.e., towards larger d-values.This is an indication of swelling when water is added to the structure.Notice that the XRPD-analysis was made on a ZBH and therefore the strongpeaks at 2 Theta=26, 35 and 38 from the porous substrate are missing.

EXAMPLE 6 Methods DSC and TGA

The DSC analysis was made on a Mettler Toledo DSC model 822. TGAanalysis was made on a Mettler Toledo TGA/SDTA 851.

DVS-analysis

A DVS analysis was made on a DVS AdVantage (Surface MeasurementSystems).

XRPD

X-Ray powder diffraction patterns were collected on a PANalytical X'PertPRO diffractometer using copper radiation equipped with PIXcel detector,automatic divergence and anti-scatter slits, soller slits and Ni-filter.The dry sample was applied to the ZBH with standard techniques for XRPD.The wet sample was analyzed by the use of porous Al₂O₃ plates toeliminate some of the solvent effects.

Microscope

Pictures were taken under microscope to compare with the Malvernresults. The dry sample was applied on a microscope slide and someMiglyol was added. The slurry sample was analysed as it was or with adrop of Miglyol.

Equipment

Slurry experiments were performed in 4 ml vials using magnetic stirring.

While the invention has been described and pointed out in detail withreference to operative embodiments thereof, it will be understood bythose skilled in the art that various changes, modifications,substitutions, and omissions can be made without departing from thespirit of the invention. It is intended therefore, that the inventionembrace those equivalents within the scope of the claims that follow.

1-20. (canceled)
 21. A method for preparing a protected linearpeptidomimetic compound of formula (IV)

wherein: R₂ and R₄ are independently hydrogen or C₁ to C₆ alkyl; R₅, R₆,R₇, R₈ are independently hydrogen or C₁ to C₆ alkyl; R₁₀ is hydrogen,methyl, t-butyl, or a protecting group; X is selected from the groupconsisting of O, NR₉, S, P, Se, C₁ to C₆ alkylene, SO, SO₂ and NH; n is0, 1, 2, 3, 4 or 5; R₁₁ and R₁₃ are independently hydrogen or aprotecting group; R₁₂ is a protecting group; W₁ and W₃ are independentlyan amino acid side chain substituent of a natural or unnatural aminoacid, less a hydrogen atom at the point of attachment to R₁₁ and R₁₃respectively; and Z is selected from the group consisting of F, Cl, Brand I; the method comprising the steps of: (a) providing a compound offormula (I)

wherein R₂, R₄, R₅, R₆, R₇, R₈, R₁₀, R₁₁, R₁₂, R₁₃, W₁, W₃, X, and nhave the meanings defined above and R₁₀ is hydrogen; (b) reacting thecompound of formula (I) with a compound of formula (II)

wherein Z and Y have the meanings defined above, to obtain a reactionmixture comprising the compound of formula (IV); (c) precipitating thecompound of formula (IV) in methyltetrahydrofuranan (MeTHF) andmethyl-t-butyl ether (MTBE).
 22. The method of claim 1, wherein (c) iscarried out at a temperature of from about 15° C. to about 30° C. 23.The method of claim 2, wherein the temperature is about 20° C.
 24. Themethod of claim 1, wherein the MeTHF and MTBE are present in a volumeratio of about one to one.
 25. The method of claim 1, wherein thecompound of formula (IV) is compound (4a):